Human FGF-21 gene and gene expression products

ABSTRACT

This invention relates to human fibroblast growth factor (hFGF-21), and to variants thereof and to polynucleotides encoding FGF-21. The invention also relates to diagnostic and therapeutic agents related to the polynucleotides and proteins, including probes and antibodies, and to methods of treating liver disease such as cirrhosis and cancer, methods of treating conditions related to thymic function, and methods of treating conditions of the testis. The invention also relates to mouse fibroblast growth factor (mFGF-21), and to variants thereof and polynucleotides encoding mFGF-21.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional PatentApplication No. 60/166,540 filed Nov. 18, 1999 and U.S. ProvisionalPatent Application No. 60/203,633 filed May 11, 2000, which areincorporated by reference herein in their entirety.

TECHNICAL FIELD

[0002] The present invention relates to nucleic acid sequences encodinga member of the fibroblast growth factor (FGF) family, and topolypeptides encoded by the nucleic acid sequence.

BACKGROUND OF THE INVENTION

[0003] The prototypic fibroblast growth factors (FGFs), FGF-1 and FGF-2,were originally isolated from brain and pituitary as mitogens forfibroblasts. However, FGF-1 and FGF-2 are widely expressed in developingand adult tissues, and are polypeptides with multiple biologicalactivities including angiogenesis, mitogenesis, cellular differentiationand repair of tissue injury (Baird, A. et al., Cancer Cells 3:239-243(1991); Burgess, W. H. et al., Annu. Rev. Biochem. 58:575-606 (1989).According to the published literature, the FGF family now consists of atleast nineteen members, FGF-1 to FGF-19. FGF-3 was identified to be acommon target for activation by the mouse mammary tumor virus (Dicksonet al., Ann. N.Y. Acad. Sci. 638:18-26 (1991); FGF-4 to FGF-6 wereidentified as oncogene products (Yoshida et al., Ann. NY Acad. Sci.638:27-37 (1991); Goldfarb et al., Ann. NY Acad. Sci 638:38-52 (1991);Coulier et al., Ann. NY Acad. Sci. 638:53-61 (1991)). FGF-10 wasidentified from rat lung by homology-based polymerase chain reaction(PCR) (Yamasaki et al., J. Biol. Chem. 271:15918-15921 (1996)). FGF-11to FGF-14 (FGF homologous factors (FHFs) 1 to 4) were identified fromhuman retina by a combination of random cDNA sequencing, data basesearches and homology-based PCR (Smallwood et al., Proc. Natl. Acad.Sci. USA 93:9850-9857 (1996)). FGF-15 was identified as a downstreamtarget of a chimeric homeodomain oncoprotein (McWhirter et al.,Development 124:3221-3232 (1997)). FGF-16, FGF-17, and FGF-18 wereidentified from rat heart and embryos by homology-based PCR,respectively (Miyake et al., Biochem. Biophys. Res. Commun. 243:148-152(1998); Hoshikawa et al., Biochem. Biophys. Res. Commun. 244:187-191(1998); Ohbayashi et al., J. Biol. Chem. 273:18161-18164 (1998)).Recently, FGF-19 was identified from human fetal brain by data basesearch (Nishimura et al., Biochim. Biophys. Acta 1444:148-151 (1999)).They have a conserved ˜120-amino acid residue core with ˜30 to 60% aminoacid identity. These FGFs also appear to play important roles in bothdeveloping and adult tissues. Thus, there is a need in the art foradditional FGF molecules having functions and activities that differfrom the known FGFs and for FGF molecules specifically expressed intissues implicated in human disease.

SUMMARY OF THE INVENTION

[0004] The present invention provides a composition comprising anisolated polynucleotide selected from the group consisting of:

[0005] (a) a polynucleotide comprising at least eight contiguousnucleotides of SEQ ID NO:1 or 3;

[0006] (b) a polynucleotide having at least 80% homology to thepolynucleotide of (a); and

[0007] (c) a polynucleotide encoding a protein expressed by apolynucleotide having the sequence of SEQ ID NO: 1 or 3.

[0008] The invention further provides for the use of the isolatedpolynucleotides or fragments thereof as diagnostic probes or as primers.

[0009] The present invention also provides a composition comprising apolypeptide, wherein said polypeptide is selected from the groupconsisting of:

[0010] (a) a polypeptide comprising at least 6 contiguous amino acidsencoded by SEQ ID NO:1 or 3;

[0011] (b) a polypeptide encoded by a polynucleotide comprising SEQ IDNO: 1 or 3; and

[0012] (c) a variant of the polypeptide of SEQ ID NO:2 or 4.

[0013] In certain preferred embodiments of the invention, thepolynucleotide is operably linked to an expression control sequence. Theinvention further provides a host cell, including bacterial, yeast,insect and mammalian cells, transformed with the polynucleotidesequence. The invention also provides full-length cDNA and full-lengthpolynucleotides corresponding to SEQ ID NO: 1 or 3.

[0014] Protein and polypeptide compositions of the invention may furthercomprise a pharmaceutically acceptable carrier. Compositions comprisingan antibody that specifically reacts with such protein or polypeptideare also provided by the present invention.

[0015] The invention also provides for the production of large amountsof otherwise minor cell populations of cells to be used for generationof cDNA libraries for the isolation of rare molecules expressed in theprecursors cells or progeny; cells produced by treatment may directlyexpress growth factors or other molecules, and conditioned media isscreened in assays for novel activities.

[0016] The invention further provides for the isolation, self-renewaland survival of mammalian stem cells and the differentiation of theirprogeny.

[0017] The invention also provides for compositions and methods ofpreventing or slowing the degeneration of or increasing the numbers ofhepatic cells, in disease states including but not limited to, cirrhosisof the liver, hepatitis, and post-surgical and post-injury tissueregeneration; of preventing or slowing degeneration of or increasing thenumbers of cells in the testes in disease states such as infertility andimpotence, and of preventing or slowing degeneration of or increasingthe numbers of cells of the thymus in disorders of the thymus and immunesystem.

[0018] The invention also provides for compositions and methods foridentifying inhibitors of FGF-21 function, useful in disease states suchas liver and testicular cancers, or leukemias, lymphomas or othercancers, and proliferative or differentiation disorders of cells derivedfrom the thymus.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1. Amino acid sequence comparison of human FGF-21 with mouseFGF-15. Asterisks indicate identical amino acid residues of thesequences.

[0020]FIG. 2. Amino acid sequence comparison of human FGF-21 and humanFGF-19. Asterisks indicate identical amino acid residues of thesequences.

[0021]FIG. 3. Expression of FGF-21 in mouse tissues.

[0022]FIG. 4. DNA sequence (SEQ ID NO:1) and amino acid sequence (SEQ IDNO:2) of mouse FGF-21.

[0023]FIG. 5. DNA sequence (SEQ ID NO:3) and amino acid sequence (SEQ IDNO:4) of human FGF-21.

[0024]FIG. 6. Alignment of the amino acid sequences of human (SEQ IDNO:4) and mouse (SEQ ID NO:2) FGF-21.

[0025]FIG. 7. FIG. 7 provides codon usage for yeast. The first field ofinformation on each line of the table contains a three-letter code foran amino acid. The second field contains an unambiguous codon for thatamino acid. The third field lists the number of occurrences of thatcodon in the genes from which the table is compiled. The fourth fieldlists the expected number of occurrences of that codon per 1,000 codonsin genes whose codon usage is identical to that compiled in the codonfrequency table. The last field contains the fraction of occurrences ofthe codon in its synonymous codon family.

[0026]FIG. 8. FIG. 8 provides codon usage for Drosophila.

[0027]FIG. 9. FIG. 9 provides codon usage for E. coli.

DETAILED DESCRIPTION OF THE INVENTION

[0028] Because of their potent activities for promoting growth,proliferation, survival and differentiation of a wide variety of cellsand tissue types, FGFs continue to be pursued as therapeutic agents fora number of different indications, including wound healing, such asmusculo-skeletal conditions, for example, bone fractures, ligament andtissue repair, tendonitis, bursitis, etc.; skin conditions, for example,bums, cuts, lacerations, bed sores, slow healing ulcers, etc.; tissueprotection, repair, and the induction of angiogenesis during myocardialinfarction and ischemia, in the treatment of neurological conditions,for example, neuro-degenerative disease and stroke, in the treatment ofeye disease, including macular degeneration, and the like.

[0029] The fibroblast growth factor (FGF) proteins identified to datebelong to a family of signaling molecules that regulate growth anddifferentiation of a variety of cell types. The significance of FGFproteins to human physiology and pathology relates in part to their keyroles in embryogenesis, in blood vessel development and growth, and inbone growth. In vitro experiments have demonstrated a role for FGF inregulating cell growth and division of endothelial cells, vascularsmooth muscle cells, fibroblasts, and cardiac and skeletal myocytes.Other members of the FGF family and their biological roles are describedin Crossley et al., Development 121:439-451 (1995); Ohuchi et al.,Development 124:2235-2244 (1997); Gemel et al., Genomics 35:253-257(1996); and Ghosh et al., Cell Growth and Differentiation 7:1425-1434(1996).

[0030] FGF proteins are also significant to human health and diseasebecause of a role in cancer cell growth. For example, FGF-8 wasidentified as an androgen-induced growth factor in breast and prostatecancer cells. (Tanaka et al., FEBS Lett. 363:226-230 (1995) and P.N.A.S.89:8928-8932 (1992)).

[0031] The role of FGF in normal development is being elucidated in partthrough studies of FGF receptors. Wilke, T. et al., Dev. Dynam.210:41-52 (1997) found that FGFR1, FGFR2, and FGFR3 transcripts werelocalized to specific regions of the head during embryonic developmentin chickens. The expression pattern correlated with areas affected byhuman FGFR mutations in Crouzon syndrome, a condition of abnormalintramembranous bone formation. Belluardo, N. et al., Jour. Comp. Neur.379:226-246 (1997) studied localization of FGFR 1, 2, and 3 mRNAs in ratbrain, and found cellular specificity in several brain regions.Furthermore, FGFR1 and FGFR2 mRNAs were expressed in astroglial reactivecells after brain lesion, supporting a role of certain FGF's in braindisease and injury. Ozawa, K. et al., Mol. Brain Res. 41:279-288 (1996)reported that FGF 1 and FGF-5 expression increased after birth, whereasFGF3, FGF-6, FGF-7, and FGF-8 genes showed higher expression in lateembryonic stages than in postnatal stages.

[0032] New members of the FGF family are described here, wherein the FGFprotein is expressed in a variety of tissues but most abundantly in theliver. A polynucleotide encoding the mouse FGF of the invention has thesequence as shown in SEQ ID NO:1. A polynucleotide encoding the humanFGF of the invention has the sequence as shown in SEQ ID NO:3. The mousepolynucleotide was identified as encoding a member of the FGF family bythe conserved regions throughout the amino acid sequence and by theregions of homology shared by the polynucleotide and genes encodingknown FGF proteins.

[0033] The inventors believe that FGF-21 is a previously unidentifiedmember of the FGF family. To date, over 19 human FGF proteins have beenidentified. In most cases, homologous proteins in other mammals,particularly mice and rats, have also been identified. The humanproteins vary to different degrees in terms of amino acid sequence,receptor specificity, tissue expression patterns, and biologicalactivity.

[0034] The present FGF-21 differs in sequence from all the FGF proteinsdescribed to date in publications. As discussed herein, the knowledgeabout the roles played by various FGF proteins continues to grow, but isby far incomplete.

[0035] The present invention adds to this knowledge by disclosing thatthe FGF of SEQ ID NO:1 is highly expressed in liver, and human FGF-21may play a role in development of and recovery from liver disease.Further, FGF-21 is also expressed in testis and thymus, and thereforemay play a role in the development or recovery from disorders oftesticular function or function of cells derived from the thymus. Theinvention therefore is based upon the identification, isolation andsequencing of a new fibroblast growth factor (FGF-21).

[0036] Isolation and Analysis of Mouse cDNA Encoding FGF-21

[0037] According to the invention, DNA encoding a novel mouse FGF hasbeen identified. The nucleotide sequence of the entire coding region wasdetermined by adaptor-ligation mediated polymerase chain reaction usingmouse embryo cDNA as a template. The nucleotide sequence of the codingregion allowed for the elucidation of the complete amino acid sequenceof the mouse FGF (210 amino acids) (FIG. 4). This protein is tentativelynamed FGF-21.

[0038] Isolation and Analysis of Human cDNA Encoding FGF-21

[0039] A human gene encoding FGF-21 was located in the 5′ flankingregion of a putative human α-fucosyltransferase gene. The cDNA encodingthe entire coding region of human FGF-21 was amplified from fetal brainCDNA by PCR using FGF-specific primers as follows: sense primer: 5′agccattgatggactcggac 3′ (SEQ ID NO:5); antisense primer: 5′tggcttcaggaagcgtagct 3′ (SEQ ID NO:6).

[0040] Expression of FGF-21 mRNA in Adult Mouse Tissues

[0041] The expression of FGF-21 MRNA was examined in adult mouse majortissues including brain, heart, lung, liver, kidney, spleen, lung,thymus, testis, muscle, skin, and small intestine by polymerase chainreaction. FGF-21 MRNA expression was detected at high levels in theliver (FIG. 3). Expression was also seen in testis and thymus. Toconfirm the expression of FGF-21 MRNA in mouse tissues, mouse tissue(A)⁺ RNA was examined by Northern blotting analysis using a ³²P-labeledrat FGF-21 cDNA probe. The results confirmed a high level of expressionin mouse liver. Expression was also seen in thymus; larger transcriptswere seen in testis tissue.

[0042] Reference to FGF-21 herein is intended to be construed to includegrowth factors of any origin which are substantially homologous to andwhich are biologically equivalent to the FGF-21 characterized anddescribed herein. Such substantially homologous growth factors may benative to any tissue or species and, similarly, biological activity canbe characterized in any of a number of biological assay systems.

[0043] The term “biologically equivalent” is intended to mean that thecompositions of the present invention are capable of demonstrating someor all of the same growth properties in a similar fashion, notnecessarily to the same degree as the FGF-21 isolated as describedherein or recombinantly produced human FGF-21 of the invention.

[0044] By “substantially homologous” it is meant that the degree ofhomology of human FGF-21 to FGF-21 from any species is greater than thatbetween FGF-21 and any previously reported member of the FGF family.

[0045] Sequence identity or percent identity is intended to mean thepercentage of same residues between two sequences, referenced to humanFGF when determining percent identity with non-human FGF-21, referencedto FGF-21 when determining percent identity with non-FGF-21 growthfactors, when the two sequences are aligned using the Clustal method(Higgins et al, Cabios 8:189-191, 1992) of multiple sequence alignmentin the Lasergene biocomputing software (DNASTAR, INC, Madison, Wis.). Inthis method, multiple alignments are carried out in a progressivemanner, in which larger and larger alignment groups are assembled usingsimilarity scores calculated from a series of pairwise alignments.Optimal sequence alignments are obtained by finding the maximumalignment score, which is the average of all scores between the separateresidues in the alignment, determined from a residue weight tablerepresenting the probability of a given amino acid change occurring intwo related proteins over a given evolutionary interval. Penalties foropening and lengthening gaps in the alignment contribute to the score.The default parameters used with this program are as follows: gappenalty for multiple alignment=10; gap length penalty for multiplealignment=10; k-tuple value in pairwise alignment=1; gap penalty inpairwise alignment=3; window value in pairwise alignment=5; diagonalssaved in pairwise alignment=5. The residue weight table used for thealignment program is PAM250 (Dayhoff et al., in Atlas of ProteinSequence and Structure, Dayhoff, Ed., NDRF, Washington, Vol. 5, suppl.3, p. 345, 1978).

[0046] Percent conservation is calculated from the above alignment byadding the percentage of identical residues to the percentage ofpositions at which the two residues represent a conservativesubstitution (defined as having a log odds value of greater than orequal to 0.3 in the PAM250 residue weight table). Conservation isreferenced to human FGF-21 when determining percent conservation withnon-human FGF-21, and referenced to FGF-21 when determining percentconservation with non-FGF-21 growth factors. Conservative amino acidchanges satisfying this requirement are: R-K; E-D, Y-F, L-M; V-I, Q-H.

[0047] The invention provides FGF-21 proteins or variants thereof havingone or more polymers covalently attached to one or more reactive aminoacid side chains. By way of example, not limitation, such polymersinclude polyethylene glycol (PEG), which can be attached to one or morefree cysteine sulfhydryl residues, thereby blocking the formation ofdisulfide bonds and aggregation when the protein is exposed to oxidizingconditions. In addition, pegylation of FGF-21 proteins and/or muteins isexpected to provide such improved properties as increased half-life,solubility, and protease resistance. FGF-21 proteins and/or muteins mayalternatively be modified by the covalent addition of polymers to freeamino groups such as the lysine epsilon or the N-terminal amino group.Preferred cysteines and lysines for covalent modification will be thosenot involved in receptor or heparin binding or in proper proteinfolding. For example, cys 27 and cys 104 may be modified. It will beapparent to one skilled in the art that the methods for assaying FGF-21biochemical and/or biological activity may be employed in order todetermine if modification of a particular amino acid residue affects theactivity of the protein as desired.

[0048] It may be advantageous to improve the stability of FGF-21 bymodifying one or more protease cleavage sites. Thus, the presentinvention provides FGF-21 variants in which one or more proteasecleavage site has been altered by, for example, substitution of one ormore amino acids at the cleavage site in order to create an FGF-21variant with improved stability. Such improved protein stability may bebeneficial during protein production and/or therapeutic use. A preferredsite is a monobasic site within two residues of a proline, such as nearresidue 160 of SEQ ID NO:4.

[0049] Suitable protease cleavage sites for modification are well knownin the art and likely will vary depending on the particular applicationcontemplated. For example, typical substitutions would includereplacement of lysines or arginines with other amino acids such asalanine. The loss of activity, such as receptor binding or heparinbinding, can be tested for as described herein.

[0050] FGF-21 can also include hybrid and modified forms of FGF-21including fusion proteins and FGF-21 fragments and hybrid and modifiedforms in which certain amino acids have been deleted or replaced andmodifications such as where one or more amino acids have been changed toa modified amino acid or unusual amino acid and modifications such asglycosylations so long as the hybrid or modified form retains thebiological activity of FGF-21. Fusion proteins can consist of the FGF-21of the invention or fragment thereof and a signal sequence of aheterologous protein to promote secretion of the protein product.

[0051] Fusion proteins comprising FGF-21 or a biologically active orantigenic fragment thereof can be produced using methods known in theart. Such fusion proteins can be used therapeutically or can be producedin order to simplify the isolation and purification procedures.Histidine residues can be incorporated to allow immobilized metalaffinity chromatography purification. Residues EQKLISEEDL contain theantigenic determinant recognized by the myc monoclonal antibody and canbe incorporated to allow myc monoclonal antibody-based affinitypurification. A thrombin cleavage site can be incorporated to allowcleavage of the molecule at a chosen site; a preferred thrombin cleavagesite consists of residues LVPRG. Purification of the molecule can befacilitated by incorporating a sequence, such as residues SAWRHPQFGG,which binds to paramagnetic streptavidin beads. Such embodiments aredescribed in WO 97/25345, which is incorporated by reference.

[0052] The invention further includes chimeric molecules between FGF-21and keratinocyte growth factor (KGF) (Reich-Slotky, R. et al., J. Biol.Chem. 270:29813-29818 (1995)). The chimeric molecule can containspecific regions or fragments of one or both of the FGF-21 and KGFmolecules, such as the FGF-21 fragments described below.

[0053] The invention also includes fragments of FGF-21. Preferredfragments of SEQ ID NO:4 and 2, respectively, include: amino acids fromabout 1 to about 209 (210 for SEQ ID NO:2); amino acids from about 2 toabout 209 (210 for SEQ ID NO:2); amino acids from about 1 to about 177;amino acids from about 40 to about 209 for SEQ ID NO:2 and amino acidsfrom about 40 to about 177. Such fragments can be prepared from theproteins by standard biochemical methods, or by expressing apolynucleotide encoding the fragment.

[0054] FGF-21, or a fragment thereof, can be produced as a fusionprotein comprising human serum albumin (HSA) or a portion thereof. Suchfusion constructs are suitable for enhancing expression of the FGF-21,or fragment thereof, in an eukaryotic host cell. Exemplary HSA portionsinclude the N-terminal polypeptide (amino acids 1-369, 1-419, andintermediate lengths starting with amino acid 1), as disclosed in U.S.Pat. No. 5,766,883, and publication WO 97/24445, incorporated byreference herein. Other chimeric polypeptides can include a HSA proteinwith FGF-21, or fragments thereof, attached to each of the C-terminaland N-terminal ends of the HSA. Such HSA constructs are disclosed inU.S. Pat. No. 5,876,969, incorporated by reference herein.

[0055] Also included with the scope of the invention are FGF-21molecules that differ from native FGF-21 by virtue of changes inbiologically active sites.

[0056] Growth factors are thought to act at specific receptors.According to the invention, FGF-21 and as yet unknown members of thisfamily of growth factors act through specific receptors having distinctdistributions as has been shown for other growth factor families.

[0057] A preferred hFGF-21 of the present invention has been identified.Also preferred is hFGF-21 prepared by recombinant DNA technology.Included within the scope of the invention are polynucleotides,including DNA and RNA, with 80% homology to SEQ ID NO:1 or SEQ ID NO:3;preferably at least 85% homology, more preferably at least 90% homology,most preferably 95% homology. Polynucleotides with 96%, 97%, 98%, and99% homology to SEQ ID NO: 1 or 3 are also included. Percent homology iscalculated using methods known in the art. A non-limiting example ofsuch a method is the Smith-Waterman homology search algorithm asimplemented in MPSRCH program (Oxford Molecular), using an affine gapsearch with a gap open penalty of 12 and a gap extension penalty of 1.

[0058] FGF-21 can also include hybrid and modified forms of FGF-21including fusion proteins and FGF-21 fragments and hybrid and modifiedforms in which certain amino acids have been deleted or replaced andmodifications such as where one or more amino acids have been changed toa modified amino acid or unusual amino acid and modifications such asglycosylations so long as the hybrid or modified form retains thebiological activity of FGF-21. By retaining the biological activity, itis meant that the ability of FGF-21 to promote the growth, survival ordifferentiation of responsive cells is preserved, although notnecessarily at the same level of potency as that of the FGF-21 isolatedas described herein or that of the recombinantly produced FGF-21.

[0059] Also included within the meaning of substantially homologous isany FGF-21 which may be isolated by virtue of cross-reactivity withantibodies to the FGF-21 described herein or whose encoding nucleotidesequences including genomic DNA, MRNA or cDNA may be isolated throughhybridization with the complementary sequence of genomic or subgenomicnucleotide sequences or cDNA of the FGF-21 herein or fragments thereof.It will also be appreciated by one skilled in the art that degenerateDNA sequences can encode human FGF-21 and these are also intended to beincluded within the present invention as are allelic variants of FGF-21.

[0060] Recombinant human FGF-21 may be made by expressing the DNAsequences encoding FGF-21 in a suitable transformed host cell. Usingmethods well known in the art, the DNA encoding FGF-21 may be linked toan expression vector, transformed into a host cell and conditionsestablished that are suitable for expression of FGF-21 by thetransformed cell.

[0061] The DNA encoding FGF-21 can be engineered to take advantage ofpreferred codon usage of host cells. Codon usage in Pseudomonasaeruginosa is described in, for example, West et al., Nucleic Acids Res.11:9323-9335 (1988). Codon usage in Saccharomyces cerevisiae isdescribed in, for example, Lloyd et al., Nucleic Acids Res. 20:5289-5295(1992). Codon preference in Corynebacteria and a comparison with E. colipreference is provided in Malubres et al., Gene 134:15-24 (1993). Codonusage in Drosophila melanogaster is described in, for example, Akashi,Genetics 136:927-935 (1994). Codon usage in yeast is also shown in FIG.7, codon usage in Drosophila is shown in FIG. 8, and codon usage for E.coli is shown in FIG. 9.

[0062] Any suitable expression vector may be employed to producerecombinant human FGF-21 such as expression vectors for use in insectcells. Baculovirus expression systems can also be employed. A preferablemethod is expression in insect cells, such as Tr5 or Sf9 cells, usingbaculovirus vector.

[0063] The present invention includes nucleic acid sequences includingsequences that encode human FGF-21. Also included within the scope ofthis invention are sequences that are substantially the same as thenucleic acid sequences encoding FGF-21. Such substantially the samesequences may, for example, be substituted with codons more readilyexpressed in a given host cell such as E. coli according to well knownand standard procedures. Such modified nucleic acid sequences areincluded within the scope of this invention.

[0064] Specific nucleic acid sequences can be modified by those skilledin the art and, thus, all nucleic acid sequences that code for the aminoacid sequences of FGF-21 can likewise be so modified. The presentinvention thus also includes nucleic acid sequence which will hybridizewith all such nucleic acid sequences, or complements of the nucleic acidsequences where appropriate, and encode a polypeptide having the cellsurvival, growth or differentiation activity of FGF-21. The presentinvention also includes nucleic acid sequences that encode polypeptidesthat have cell survival promoting activity and that are recognized byantibodies that bind to FGF-21. Preferred methods and epitopes forraising antibodies are described in Example 4.

[0065] The present invention also encompasses vectors comprisingexpression regulatory elements operably linked to any of the nucleicacid sequences included within the scope of the invention. Thisinvention also includes host cells of any variety that have beentransformed with vectors comprising expression regulatory elementsoperably linked to any of the nucleic acid sequences included within thescope of the present invention.

[0066] Methods are also provided herein for producing FGF-21.Preparation can be by isolation from conditioned medium from a varietyof cell types so long as the cell type produces FGF-21. A second andpreferred method involves utilization of recombinant methods byisolating or obtaining a nucleic acid sequence encoding FGF-21, cloningthe sequence along with appropriate regulatory sequences into suitablevectors and cell types, and expressing the sequence to produce FGF-21.

[0067] Although FGF-21 has been described on the basis of its highexpression level in liver, this factor may act on other cell types aswell. It is likely that FGF-21 will act on non-liver cells to promotetheir survival, growth, differentiation state or function. Thisexpectation is based upon the activity of known growth factors. Membersof the FGF family act on many cell types of different function andembryologic origin, even when their expression is limited to one or afew tissues.

[0068] The inventors herein have identified that FGF-21 is expressed ata higher level in liver. This suggests a role for FGF-21 in, forexample, precancerous lesions, hepatoma, cirrhosis, repair, frominflammatory diseases, trauma or other types of injury, and otherdiseases of the liver. Further, FGF-21 is also expressed in thymus andtestis. This suggests a role for FGF-21 in, for example, infertility,control of testosterone production, cancer of the testis or associatedcells, and other disorders of the testis, and in disorders of cells suchas immune cells derived from the thymus, for example, autoimmunedisorders, leukemias and lymphomas, immune deficiency states, and thelike.

[0069] The present invention also includes therapeutic or pharmaceuticalcompositions comprising FGF-21 in an effective amount for treatingpatients with liver, testis or thymic disease, and a method comprisingadministering a therapeutically effective amount of FGF-21. Thesecompositions and methods are useful for treating a number of diseases.The compositions and methods herein can also be useful to preventdegeneration and/or promote survival in other non-liver tissues as well,such as promoting angiogenesis, neuronal survival, wound healing, andthe like. One skilled in the art can readily use a variety of assaysknown in the art to determine whether FGF-21 would be useful inpromoting survival or functioning in a particular cell type. Promotionof neuronal survival is useful in the treatment of nervous systemdiseases and conditions, including Parkinson's disease, Alzheimersdisease, traumatic injury to nerves, and degenerative disease of thenervous system.

[0070] In certain circumstances, it may be desirable to modulate ordecrease the amount of FGF-21 expressed. Thus, in another aspect of thepresent invention, FGF-21 anti-sense oligonucleotides can be made and amethod utilized for diminishing the level of expression of FGF-21 by acell comprising administering one or more FGF-21 anti-senseoligonucleotides. By FGF-21 anti-sense oligonucleotides reference ismade to oligonucleotides that have a nucleotide sequence that interactsthrough base pairing with a specific complementary nucleic acid sequenceinvolved in the expression of FGF-21 such that the expression of FGF-21is reduced. Preferably, the specific nucleic acid sequence involved inthe expression of FGF-21 is a genomic DNA molecule or MRNA molecule thatencodes FGF-21. This genomic DNA molecule can comprise regulatoryregions of the FGF-21 gene, or the coding sequence for mature FGF-21protein. The term complementary to a nucleotide sequence in the contextof FGF-21 antisense oligonucleotides and methods therefor meanssufficiently complementary to such a sequence as to allow hybridizationto that sequence in a cell, i.e., under physiological conditions. TheFGF-21 antisense oligonucleotides preferably comprise a sequencecontaining from about 8 to about 100 nucleotides and more preferably theFGF-21 antisense oligonucleotides comprise from about 15 to about 30nucleotides. The FGF-21 antisense oligonucleotides can also contain avariety of modifications that confer resistance to nucleolyticdegradation such as, for example, modified intemucleoside linages(Uhlmann and Peyman, Chemical Reviews 90:543-548 1990; Schneider andBanner, Tetrahedron Lett. 31:335, 1990 which are incorporated byreference), modified nucleic acid bases and/or sugars and the like.

[0071] The therapeutic or pharmaceutical compositions of the presentinvention can be administered by any suitable route known in the artincluding for example intravenous, subcutaneous, intramuscular,transdermal, intrathecal or intracerebral. Administration can be eitherrapid as by injection or over a period of time as by slow infusion oradministration of slow release formulation.

[0072] FGF-21 can also be linked or conjugated with agents that providedesirable pharmaceutical or pharmacodynamic properties. For example,FGF-21 can be coupled to any substance known in the art to promotepenetration or transport across the blood-brain barrier such as anantibody to the transferring receptor, and administered by intravenousinjection (see, for example, Friden et al., Science 259:373-377, 1993which is incorporated by reference). Furthermore, FGF-21 can be stablylinked to a polymer such as polyethylene glycol to obtain desirableproperties of solubility, stability, half-life and otherpharmaceutically advantageous properties. (See, for example, Davis etal., Enzyme Eng. 4:169-73, 1978; Burniham, Am. J. Hosp. Pharm.51:210-218, 1994 which are incorporated by reference.)

[0073] The compositions are usually employed in the form ofpharmaceutical preparations. Such preparations are made in a manner wellknown in the pharmaceutical art. One preferred preparation utilizes avehicle of physiological saline solution, but it is contemplated thatother pharmaceutically acceptable carriers such as physiologicalconcentrations of other non-toxic salts, five percent aqueous glucosesolution, sterile water or the like may also be used. It may also bedesirable that a suitable buffer be present in the composition. Suchsolutions can, if desired, be lyophilized and stored in a sterileampoule ready for reconstitution by the addition of sterile water forready injection. The primary solvent can be aqueous or alternativelynon-aqueous. FGF-21 can also be incorporated into a solid or semi-solidbiologically compatible matrix which can be implanted into tissuesrequiring treatment.

[0074] The carrier can also contain other pharmaceutically-acceptableexcipients for modifying or maintaining the pH, osmolarity, viscosity,clarity, color, sterility, stability, rate of dissolution, or odor ofthe formulation. Similarly, the carrier may contain still otherpharmaceutically-acceptable excipients for modifying or maintainingrelease or absorption or penetration across the blood-brain barrier.Such excipients are those substances usually and customarily employed toformulate dosages for parenteral administration in either unit dosage ormulti-dose form or for direct infusion into the cerebrospinal fluid bycontinuous or periodic infusion.

[0075] Dose administration can be repeated depending upon thepharmacokinetic parameters of the dosage formulation and the route ofadministration used.

[0076] It is also contemplated that certain formulations containingFGF-21 are to be administered orally. Such formulations are preferablyencapsulated and formulated with suitable carriers in solid dosageforms. Some examples of suitable carriers, excipients, and diluentsinclude lactose, dextrose, sucrose, sorbitol, mannitol, starches, gumacacia, calcium phosphate, alginates, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium,stearate, water, mineral oil, and the like. The formulations canadditionally include lubricating agents, wetting agents, emulsifying andsuspending agents, preserving agents, sweetening agents or flavoringagents. The compositions may be formulated so as to provide rapid,sustained, or delayed release of the active ingredients afteradministration to the patient by employing procedures well known in theart. The formulations can also contain substances that diminishproteolytic degradation and promote absorption such as, for example,surface active agents.

[0077] Depending on the treatment regimen contemplated, it may bedesired to control the rate of release of FGF-21 protein or variantthereof to provide long-term treatment while minimizing the frequency ofadministration. Such treatment regimens may be desired, for example,where the FGF-21 protein is found to be relatively unstable such thatthe localized concentration of active protein is at an efficacious levelfor an insufficient period of time. Thus, for example, for certaindiseases, it may not be desired or practical to perform repeated andfrequent injections. The major advantages of such sustained releasesystems include targeted local delivery of drugs at a constant rate,less drug required to treat the disease state, minimization of possibleside effects, and enhanced efficacy of treatment. Also, these forms ofdelivery systems are capable of protecting drugs that are unstable invivo and that would normally require a frequent dosing interval. Undersuch circumstances, sustained release may be achieved by one of themethods readily available in the art such as the encapsulation of FGF-21conjugated heparin-Sepharose beads to form heparin-alginate microspheresor the preparation of FGF-21 PLG microspheres.

[0078] Heparin-alginate microspheres have been successfully employed forthe delivery of Basic Fibroblast Growth Factor to tissue (Lopez et al.,Journal of Pharmacology and Experimental Therapeutics 282(1):385-390(1997)). Similarly, Alginate/heparin-Sepharose microspheres and filmshave been used as drug carriers to control the release of a basicFGF-saponin conjugate in order to control its release in small doses.Addition of heparin to solutions of bFGF prevents losses in activitythat accompany changes in pH or elevation in temperature. See, forexample, Gospodarowicz et al., J. Cell. Physiol. 128:475-484 (1986).

[0079] Binding of FGF-21 to heparin may be employed in order to enhanceits stability either during in vivo expression or administration or invitro during various stages of protein purification. Thus, by thepresent invention, heparin may be added to a solution of FGF-21 and theactivity assayed by the methods disclosed herein.

[0080] FGF-21 bound heparin-Sepharose beads may be encapsulated intocalcium alginate microspheres to permit the controlled release of theheparin-stabilized FGF-21 protein. For example, microspheres may beconstructed by dropping a mixed solution of sodium alginate with FGF-21bound heparin-Sepharose beads into a hardening solution of calciumchloride. Spheres are formed instantaneously as the mixture enters thehardening solution. The size of the microsphere may be adjusted bypassing the FGF-21 bound heparin-Sepharose beads through a cylinder ofreduced cross-sectional area such as through a hypodermic needle.

[0081] Encapsulation efficiency may be determined by comparing theamount of encapsulated growth factor with that initially present insolution. For example, the FGF-21 may be stripped from theheparin-Sepharose beads with a solution of 3 M NaCl and functionalactivity assays may be performed.

[0082] The specific dose is calculated according to the approximate bodyweight or body surface area of the patient or the volume of body spaceto be occupied. The dose will also be calculated dependent upon theparticular route of administration selected. Further refinement of thecalculations necessary to determine the appropriate dosage for treatmentis routinely made by those of ordinary skill in the art. Suchcalculations can be made without undue experimentation by one skilled inthe art in light of the activity disclosed herein in assay preparationsof target cells. Exact dosages are determined in conjunction withstandard dose-response studies. It will be understood that the amount ofthe composition actually administered will be determined by apractitioner, in the light of the relevant circumstances including thecondition or conditions to be treated, the choice of composition to beadministered, the age, weight, and response of the individual patient,the severity of the patient's symptoms, and the chosen route ofadministration.

[0083] In one embodiment of this invention, FGF-21 may betherapeutically administered by implanting into patients vectors orcells capable of producing a biologically-active form of FGF-21 or aprecursor of FGF-21, i.e., a molecule that can be readily converted to abiological-active form of FGF-21 by the body. In one approach cells thatsecrete FGF-21 may be encapsulated into semipermeable membranes forimplantation into a patient. The cells can be cells that normallyexpress FGF-21 or a precursor thereof or the cells can be transformed toexpress FGF-21 or a precursor thereof. It is preferred that the cell beof human origin and that the FGF-21 be human FGF-21 when the patient ishuman. However, the formulations and methods herein can be used forveterinary as well as human applications and the term “patient” as usedherein is intended to include human and veterinary patients.

[0084] Cells can be grown ex vivo for use in transplantation orengraftment into patients (Muench et al., Leuk. & Lymph. 16:1-11, 1994which is incorporated by reference). In another embodiment of thepresent invention, FGF-21 is used to promote the ex vivo expansion of acells for transplantation or engraftment. Current methods have usedbioreactor culture systems containing factors such as erythropoietin,colony stimulating factors, stem cell factor, and interleukins to expandhematopoietic progenitor cells for erythrocytes, monocytes, neutrophils,and lymphocytes (Verfaillie, Stem Cells 12:466-476, 1994 which isincorporated by reference). These stem cells can be isolated from themarrow of human donors, from human peripheral blood, or from umbilicalcord blood cells. The expanded blood cells are used to treat patientswho lack these cells as a result of specific disease conditions or as aresult of high dose chemotherapy for treatment of malignancy (George,Stem Cells 12(Suppl 1):249-255, 1994 which is incorporated byreference). In the case of cell transplant after chemotherapy,autologous transplants can be performed by removing bone marrow cellsbefore chemotherapy, expanding the cells ex vivo using methods that alsofunction to purge malignant cells, and transplanting the expanded cellsback into the patient following chemotherapy (for review, see Rummel andVan Zant, J. Hematotherapy 3:213-218, 1994 which is incorporated byreference). Since FGF-21 is expressed in liver cells, it is believedthat FGF-21 can function to prevent or slow the progression of cirrhosischanges in liver cells, and to promote hepatic cell regeneration afterinjury or after surgical removal of part of the liver due to disease.

[0085] In a number of circumstances it would be desirable to determinethe levels of FGF-21 in a patient. The identification of FGF-21 alongwith the present report showing expression of FGF-21 provides the basisfor the conclusion that the presence of FGF-21 serves a normalphysiological function related to cell growth and survival. Endogenouslyproduced FGF-21 may also play a role in certain disease conditions.

[0086] Given that FGF-21 is expressed in liver, thymic and testiculartissue, it is likely that the level of FGF-21 may be altered in avariety of conditions and that quantification of FGF-21 levels wouldprovide clinically useful information. Furthermore, in the treatment ofdegenerative conditions, altered physiological function or in recoveryfrom injury to the liver, testis or thymic cells, compositionscontaining FGF-21 can be administered and it would likely be desirableto achieve certain target levels of FGF-21 in sera or in any desiredtissue compartment. It would, therefore, be advantageous to be able tomonitor the levels of FGF-21 in a patient. Accordingly, the presentinvention also provides methods for detecting the presence of FGF-21 ina sample from a patient.

[0087] The term “detection” as used herein in the context of detectingthe presence of FGF-21 in a patient is intended to include determiningthe amount of FGF-21 or the ability to express an amount of FGF-21 in apatient, distinguishing FGF-21 from other growth factors, the estimationof prognosis in terms of probable outcome of a degenerative disease andprospect for recovery, monitoring the FGF-21 levels over a period oftime as a measure of status of the condition, and monitoring FGF-21levels for determining a preferred therapeutic regimen for the patient.

[0088] To detect the presence of FGF-21 in a patient, a sample isobtained from the patient. The sample can be a tissue biopsy sample or asample of blood, plasma, serum, CSF or the like. FGF-21 is expressed inliver tissues, as discussed in Example 2. Samples for detecting FGF-21can be taken from this tissue. When assessing the levels of FGF-21 inthe liver, thymus or testis, a preferred sample is a sample taken fromthese tissues or from veins draining these tissues.

[0089] In some instances it is desirable to determine whether the FGF-21gene is intact in the patient or in a tissue or cell line within thepatient. By an intact FGF-21 gene it is meant that there are noalterations in the gene such as point mutations, deletions, insertions,chromosomal breakage, chromosomal rearrangements and the like whereinsuch alteration might alter production of FGF-21 or alter its biologicalactivity, stability or the like to lead to disease processes orsusceptibility to cellular degenerative conditions. Thus, in oneembodiment of the present invention a method is provided for detectingand characterizing any alterations in the FGF-21 gene. The methodcomprises providing an oligonucleotide that contains the FGF-21 cDNA,genomic DNA or a fragment thereof or a derivative thereof. By aderivative of an oligonucleotide, it is meant that the derivedoligonucleotide is substantially the same as the sequence from which itis derived in that the derived sequence has sufficient sequencecomplementarily to the sequence from which it is derived to hybridize tothe FGF-21 gene. The derived nucleotide sequence is not necessarilyphysically derived from the nucleotide sequence, but may be generated inany manner including for example, chemical synthesis or DNA replicationor reverse transcription or transcription.

[0090] Typically, patient genomic DNA is isolated from a cell samplefrom the patient and digested with one or more restriction endonucleasessuch as, for example, TaqI and AluI. Using the Southern blot protocol,which is well known in the art, this assay determines whether a patientor a particular tissue in a patient has an intact FGF-21 gene or anFGF-21 gene abnormality.

[0091] Hybridization to an FGF-21 gene would involve denaturing thechromosomal DNA to obtain a single-stranded DNA; contacting thesingle-stranded DNA with a gene probe associated with the FGF-21 genesequence; and identifying the hybridized DNA-probe to detect chromosomalDNA containing at least a portion of a human FGF-21 gene.

[0092] The term “probe” as used herein refers to a structure comprisedof a polynucleotide that forms a hybrid structure with a targetsequence, due to complementarity of probe sequence with a sequence inthe target region. Oligomers suitable for use as probes may contain aminimum of about 8-12 contiguous nucleotides which are complementary tothe targeted sequence and preferably a minimum of about 20.

[0093] The FGF-21 gene probes of the present invention can be DNA or RNAoligonucleotides and can be made by any method known in the art such as,for example, excision, transcription or chemical synthesis. Probes maybe labeled with any detectable label known in the art such as, forexample, radioactive or fluorescent labels or enzymatic marker. Labelingof the probe can be accomplished by any method known in the art such asby PCR, random priming, end labeling, nick translation or the like. Oneskilled in the art will also recognize that other methods not employinga labeled probe can be used to determine the hybridization. Examples ofmethods that can be used for detecting hybridization include Southernblotting, fluorescence in situ hybridization, and single-strandconformation polymorphism with PCR amplification.

[0094] Hybridization is typically carried out at 25°-45° C., morepreferably at 32°-40° C. and more preferably at 37°-38° C. The timerequired for hybridization is from about 0.25 to about 96 hours, morepreferably from about one to about 72 hours, and most preferably fromabout 4 to about 24 hours.

[0095] FGF-21 gene abnormalities can also be detected by using the PCRmethod and primers that flank or lie within the FGF-21 gene. The PCRmethod is well known in the art. Briefly, this method is performed usingtwo oligonucleotide primers which are capable of hybridizing to thenucleic acid sequences flanking a target sequence that lies within anFGF-21 gene and amplifying the target sequence. The terms“oligonucleotide primer” as used herein refers to a short strand of DNAor RNA ranging in length from about 8 to about 30 bases. The upstreamand downstream primers are typically from about 20 to about 30 basepairs in length and hybridize to the flanking regions for replication ofthe nucleotide sequence. The polymerization is catalyzed by aDNA-polymerase in the presence of deoxynucleotide triphosphates ornucleotide analogs to produce double-stranded DNA molecules. The doublestrands are then separated by any denaturing method including physical,chemical or enzymatic. Commonly, the method of physical denaturation isused involving heating the nucleic acid, typically to temperatures fromabout 80° C. to 105° C. for times ranging from about 1 to about 10minutes. The process is repeated for the desired number of cycles.

[0096] The primers are selected to be substantially complementary to thestrand of DNA being amplified. Therefore, the primers need not reflectthe exact sequence of the template, but must be sufficientlycomplementary to selectively hybridize with the strand being amplified.

[0097] After PCR amplification, the DNA sequence comprising FGF-21 orpre-pro FGF-21 or a fragment thereof is then directly sequenced andanalyzed by comparison of the sequence with the sequences disclosedherein to identify alterations which might change activity or expressionlevels or the like.

[0098] In another embodiment, a method for detecting FGF-21 is providedbased upon an analysis of tissue expressing the FGF-21 gene. Certaintissues such as those identified below in Example 2 have been found toexpress the FGF-21 gene. The method comprises hybridizing apolynucleotide to mRNA from a sample of tissues that normally expressthe FGF-21 gene. The sample is obtained from a patient suspected ofhaving an abnormality in the FGF-21 gene or in the FGF-21 gene ofparticular cells.

[0099] To detect the presence of MRNA encoding FGF-21 protein, a sampleis obtained from a patient. The sample can be from blood or from atissue biopsy sample. The sample may be treated to extract the nucleicacids contained therein. The resulting nucleic acid from the sample issubjected to gel electrophoresis or other size separation techniques.

[0100] The MRNA of the sample is contacted with a DNA sequence servingas a probe to form hybrid duplexes. The use of a labeled probes asdiscussed above allows detection of the resulting duplex.

[0101] When using the cDNA encoding FGF-21 protein or a derivative ofthe cDNA as a probe, high stringency conditions can be used in order toprevent false positives, that is the hybridization and apparentdetection of FGF-21 nucleotide sequences when in fact an intact andfunctioning FGF-21 gene is not present. When using sequences derivedfrom the FGF-21 cDNA, less stringent conditions could be used, however,this would be a less preferred approach because of the likelihood offalse positives. The stringency of hybridization is determined by anumber of factors during hybridization and during the washing procedure,including temperature, ionic strength, length of time and concentrationof formamide. These factors are outlined in, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed. (1989) ColdSpring Harbor Press, Cold Spring Harbor, N.Y.

[0102] In order to increase the sensitivity of the detection in a sampleof MRNA encoding the FGF-21 protein, the technique of reversetranscription/polymerization chain reaction (RT/PCR) can be used toamplify cDNA transcribed from mRNA encoding the FGF-21 protein. Themethod of RT/PCR is well known in the art, and can be performed asfollows. Total cellular RNA is isolated by, for example, the standardguanidium isothiocyanate method and the total RNA is reversetranscribed. The reverse transcription method involves synthesis of DNAon a template of RNA using a reverse transcriptase enzyme and a 3′ endprimer. Typically, the primer contains an oligo(dT) sequence. The cDNAthus produced is then amplified using the PCR method and FGF-21 specificprimers. (Belyavsky et al., Nucl. Acid Res. 17:2919-2932, 1989; Krug andBerger, Methods in Enzymology, 152:316-325, Academic Press, NY, 1987which are incorporated by reference).

[0103] The polymerase chain reaction method is performed as describedabove using two oligonucleotide primers that are substantiallycomplementary to the two flanking regions of the DNA segment to beamplified.

[0104] Following amplification, the PCR product is then electrophoresedand detected by ethidium bromide staining or by phosphoimaging.

[0105] The present invention further provides for methods to detect thepresence of the FGF-21 protein in a sample obtained from a patient. Anymethod known in the art for detecting proteins can be used. Such methodsinclude, but are not limited to immunodiffusion, immunoelectrophoresis,immunochemical methods, binder-ligand assays, immunohistochemicaltechniques, agglutination and complement assays. (for example, see Basicand Clinical Immunology, 217-262, Sites and Terr, eds., Appleton &Lange, Norwalk, Conn., 1991 which is incorporated by reference).Preferred are binder-ligand immunoassay methods including reactingantibodies with an epitope or epitopes of the FGF-21 protein andcompetitively displacing a labeled FGF-21 protein or derivative thereof.Preferred antibodies are prepared according to Example 4.

[0106] As used herein, a derivative of the FGF-21 protein is intended toinclude a polypeptide in which certain amino acids have been deleted orreplaced or changed to modified or unusual amino acids wherein theFGF-21 derivative is biologically equivalent to FGF-21 and wherein thepolypeptide derivative cross-reacts with antibodies raised against theFGF-21 protein. By cross-reaction it is meant that an antibody reactswith an antigen other than the one that induced its formation.

[0107] Numerous competitive and non-competitive protein bindingimmunoassays are well known in the art. Antibodies employed in suchassays may be unlabeled, for example as used in agglutination tests, orlabeled for use in a wide variety of assay methods. Labels that can beused include radionuclides, enzymes, fluorescers, chemiluminescers,enzyme substrates or co-factors, enzyme inhibitors, particles, dyes andthe like for use in radioimmunoassay (RIA), enzyme immunoassays, e.g.,enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassays andthe like.

[0108] Polyclonal or monoclonal antibodies to the FGF-21 protein or anepitope thereof can be made for use in immunoassays by any of a numberof methods known in the art. By epitope reference is made to anantigenic determinant of a polypeptide. An epitope could comprise 3amino acids in a spatial conformation which is unique to the epitope.Generally an epitope consists of at least 5 such amino acids. Methods ofdetermining the spatial conformation of amino acids are known in theart, and include, for example, x-ray crystallography and 2 dimensionalnuclear magnetic resonance.

[0109] One approach for preparing antibodies to a protein is theselection and preparation of an amino acid sequence of all or part ofthe protein, chemically synthesizing the sequence and injecting it intoan appropriate animal, usually a rabbit or a mouse (see Example 4).

[0110] Oligopeptides can be selected as candidates for the production ofan antibody to the FGF-21 protein based upon the oligopeptides lying inhydrophilic regions, which are thus likely to be exposed in the matureprotein. Preferred oligopeptides are RQRYLYTDDAQQTEAH (residues 46-61 ofSEQ NO:4) and HLPGNKSPHRDPAPR (residues 146-160 of SEQ ID NO:4).Additional oligopeptides can be determined using, for example, theAntigenicity Index of Welling, G.W. et al., FEBS Lett. 188:215-218,1985, incorporated herein by reference.

[0111] Antibodies to FGF-21 can also be raised against oligopeptidesthat include one or more of the conserved regions identified herein suchthat the antibody can cross-react with other family members. Suchantibodies can be used to identify and isolate the other family members.

[0112] Methods for preparation of the FGF-21 protein or an epitopethereof include, but are not limited to chemical synthesis, recombinantDNA techniques or isolation from biological samples. Chemical synthesisof a peptide can be performed, for example, by the classical Merrifeldmethod of solid phase peptide synthesis (Merrifeld, J. Am. Chem. Soc.85:2149, 1963 which is incorporated by reference) or the FMOC strategyon a Rapid Automated Multiple Peptide Synthesis system (E. I. du Pont deNemours Company, Wilmington, Del.) (Caprino and Han, J. Org. Chem.37:3404, 1972 which is incorporated by reference).

[0113] Polyclonal antibodies can be prepared by immunizing rabbits orother animals by injecting antigen followed by subsequent boosts atappropriate intervals. The animals are bled and sera assayed againstpurified FGF-21 protein usually by ELISA or by bioassay based upon theability to block the action of FGF-21 on liver or other cells. Whenusing avian species, e.g., chicken, turkey and the like, the antibodycan be isolated from the yolk of the egg. Monoclonal antibodies can beprepared after the method of Milstein and Kohler by fusing splenocytesfrom immunized mice with continuously replicating tumor cells such asmyeloma or lymphoma cells. (Milstein and Kohler, Nature 256:495-497,1975; Gulfre and Milstein, Methods in Enzymology: ImmunochemicalTechniques 73:1-46, Langone and Banatis eds., Academic Press, 1981 whichare incorporated by reference). The hybridoma cells so formed are thencloned by limiting dilution methods and supernates assayed for antibodyproduction by ELISA, RIA or bioassay.

[0114] The unique ability of antibodies to recognize and specificallybind to target proteins provides an approach for treating anoverexpression of the protein. Thus, another aspect of the presentinvention provides for a method for preventing or treating diseasesinvolving overexpression of the FGF-21 protein by treatment of a patientwith specific antibodies to the FGF-21 protein.

[0115] Specific antibodies, either polyclonal or monoclonal, to theFGF-21 protein can be produced by any suitable method known in the artas discussed above. For example, murine or human monoclonal antibodiescan be produced by hybridoma technology or, alternatively, the FGF-21protein, or an immunologically active fragment thereof, or ananti-idiotypic antibody, or fragment thereof can be administered to ananimal to elicit the production of antibodies capable of recognizing andbinding to the FGF-21 protein. Such antibodies can be from any class ofantibodies including, but not limited to IgG, IgA, IgM, IgD, and IgE orin the case of avian species, IgY and from any subclass of antibodies.

[0116] Polypeptides encoded by the instant polynucleotides andcorresponding full-length genes can be used to screen peptide libraries,protein libraries, small molecule libraries, and phage displaylibraries, and other known methods, to identify analogs or antagonists.

[0117] Native FGF polypeptides may play a role in cancer. For example,FGF family members can induce marked morphological transformation of NIH3T3 cells, and exhibit strong tumorigenicity in nude mice. Angiogenicactivity has been exhibited by FGF family members. Thus, inhibitors ofFGF can be used to treat cancer, such as prostate cancer.

[0118] A library of peptides may be synthesized following the methodsdisclosed in U.S. Pat. No. 5,010,175, and in PCT No. WO 91/17823. Asdescribed below in brief, a 5 mixture of peptides is prepared, which isthen screened to identify the peptides exhibiting the desired signaltransduction and receptor binding activity. According to the method ofthe '175 patent, a suitable peptide synthesis support (e.g., a resin) iscoupled to a mixture of appropriately protected, activated amino acids.The concentration of each amino acid in the reaction mixture is balancedor adjusted in inverse proportion to its coupling reaction rate so thatthe product is an equimolar mixture of amino acids coupled to thestarting resin. The bound amino acids are then deprotected, and reactedwith another balanced amino acid mixture to form an equimolar mixture ofall possible dipeptides. This process is repeated until a mixture ofpeptides of the desired length (e.g., hexamers) is formed. Note that oneneed not include all amino acids in each step: one may include only oneor two amino acids in some steps (e.g., where it is known that aparticular amino acid is essential in a given position), thus reducingthe complexity of the mixture. After the synthesis of the peptidelibrary is completed, the mixture of peptides is screened for binding tothe selected polypeptide. The peptides are then tested for their abilityto inhibit or enhance activity. Peptides exhibiting the desired activityare then isolated and sequenced.

[0119] The method described in PCT No. WO 91/17823 is similar. However,instead of reacting the synthesis resin with a mixture of activatedamino acids, the resin is divided into twenty equal portions (or into anumber of portions corresponding to the number of different amino acidsto be added in that step), and each amino acid is coupled individuallyto its portion of resin. The resin portions are then combined, mixed,and again divided into a number of equal portions for reaction with thesecond amino acid. In this manner, each reaction may be easily driven tocompletion. Additionally, one may maintain separate “subpools” bytreating portions in parallel, rather than combining all resins at eachstep. This simplifies the process of determining which peptides areresponsible for any observed receptor binding or signal transductionactivity.

[0120] In such cases, the subpools containing, e.g., 1-2,000 candidateseach are exposed to one or more polypeptides of the invention. Eachsubpool that produces a positive result is then resynthesized as a groupof smaller subpools (sub-subpools) containing, e.g., 20-100 candidates,and reassayed. Positive sub-subpools may be resynthesized as individualcompounds, and assayed finally to determine the peptides that exhibit ahigh binding constant. These peptides can be tested for their ability toinhibit or enhance the native activity. The methods described in PCT No.WO 91/7823 and U.S. Pat. No. 5,194,392 (herein incorporated byreference) enable the preparation of such pools and subpools byautomated techniques in parallel, such that all synthesis andresynthesis may be performed in a matter of days.

[0121] Peptide agonists or antagonists are screened using any availablemethod, such as signal transduction, antibody binding, receptor bindingand mitogenic assays. The assay conditions ideally should resemble theconditions under which the native activity is exhibited in vivo, thatis, under physiologic pH, temperature, and ionic strength. Suitableagonists or antagonists will exhibit strong inhibition or enhancement ofthe native activity at concentrations that do not cause toxic sideeffects in the subject. Agonists or antagonists that compete for bindingto the native polypeptide may require concentrations equal to or greaterthan the native concentration, while inhibitors capable of bindingirreversibly to the polypeptide may be added in concentrations on theorder of the native concentration.

[0122] The availability of hFGF-21 and mFGF-21 allows for theidentification of small molecules and low molecular weight compoundsthat inhibit the binding of FGF-21 to its receptor, through routineapplication of high-throughput screening methods (HTS). HTS methodsgenerally refer to technologies that permit the rapid assaying of leadcompounds for therapeutic potential. HTS techniques employ robotichandling of test materials, detection of positive signals, andinterpretation of data. Lead compounds may be identified via theincorporation of radioactivity or through optical assays that rely onabsorbance, fluorescence or luminescence as read-outs. Gonzalez, J. E.et al., (1998) Curr. Opin. Biotech. 9:624-631. Assays for detectinginteraction between an FGF molecule and FGF receptor are described in,for example, Blunt, A. G. et al., (1997) J. Biol. Chem. 272:3733 -3738,and such assays can be adapted for determining if a candidate moleculecan inhibit the interaction between FGF-21 and its receptor.

[0123] Model systems are available that can be adapted for use in highthroughput screening for compounds that inhibit the interaction ofFGF-21 with its receptor, for example by competing with FGF-21 forreceptor binding. Sarubbi et al., (1996) Anal. Biochem. 237:70-75describe cell-free, non-isotopic assays for identifying molecules thatcompete with natural ligands for binding to the active site of IL-1receptor. Martens, C. et al., (1999) Anal. Biochem. 273:20-31 describe ageneric particle-based nonradioactive method in which a labeled ligandbinds to its receptor immobilized on a particle; label on the particledecreases in the presence of a molecule that competes with the labeledligand for receptor binding.

[0124] The therapeutic FGF-21 polynucleotides and polypeptides of thepresent invention may be utilized in gene delivery vehicles. The genedelivery vehicle may be of viral or non-viral origin (see generally,Jolly, Cancer Gene Therapy 1:51-64 (1994); Kimura, Human Gene Therapy5:845-852 (1994); Connelly, Human Gene Therapy 1:185-193 (1995); andKaplitt, Nature Genetics 6:148-153 (1994)). Gene therapy vehicles fordelivery of constructs including a coding sequence of a therapeutic ofthe invention can be administered either locally or systemically. Theseconstructs can utilize viral or non-viral vector approaches. Expressionof such coding sequences can be induced using endogenous mammalian orheterologous promoters. Expression of the coding sequence can be eitherconstitutive or regulated.

[0125] The present invention can employ recombinant retroviruses whichare constructed to carry or express a selected nucleic acid molecule ofinterest. Retrovirus vectors that can be employed include thosedescribed in EP 0 415 731; WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; Vile andHart, Cancer Res. 53:3860-3864 (1993); Vile and Hart, Cancer Res.53:962-967 (1993); Ram et al., Cancer Res. 53:83-88 (1993); Takamiya etal., J. Neurosci. Res. 33:493-503 (1992); Baba et al., J. Neurosurg.79:729-735 (1993); U.S. Pat. No. 4,777,127; GB Patent No. 2,200,651; andEP 0 345 242. Preferred recombinant retroviruses include those describedin WO 91/02805.

[0126] Packaging cell lines suitable for use with the above-describedretroviral vector constructs may be readily prepared (see PCTpublications WO 95/30763 and WO 92/05266), and used to create producercell lines (also termed vector cell lines) for the production ofrecombinant vector particles. Within particularly preferred embodimentsof the invention, packaging cell lines are made from human (such asHT1080 cells) or mink parent cell lines, thereby allowing production ofrecombinant retroviruses that can survive inactivation in human serum.

[0127] The present invention also employs alphavirus-based vectors thatcan function as gene delivery vehicles. Such vectors can be constructedfrom a wide variety of alphaviruses, including, for example, Sindbisvirus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), RossRiver virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equineencephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCCVR-532). Representative examples of such vector systems include thosedescribed in U.S. Pat. Nos. 5,091,309; 5,217,879; and 5,185,440; and PCTPublication Nos. WO 92/10578; WO 94/21792; WO 95/27069; WO 95/27044; andWO 95/07994.

[0128] Gene delivery vehicles of the present invention can also employparvovirus such as adeno-associated virus (AAV) vectors. Representativeexamples include the AAV 20 vectors disclosed by Srivastava in WO93/09239, Samulski et al., J. Vir. 63:3822-3828 (1989); Mendelson etal., Virol. 166:154-165 (1988); and Flotte et al., P.N.A.S.90:10613-10617 (1993).

[0129] Representative examples of adenoviral vectors include thosedescribed by Berkner, Biotechniques 6:616-627 (Biotechniques); Rosenfeldet al., Science 252:431-434 (1991); WO 93/19191; Kolls et al., P.N.A.S.:215-219 (1994); Kass-Eisleret al., P.N.A.S. 90:11498-11502 (1993);Guzman et al., Circulation 88:2838-2848 (1993); Guzman et al., Cir. Res.73:1202-1207 (1993); Zabner et al., Cell 75:207-216 (1993); Li et al.,Hum. Gene Ther. 4:403-409 (1993); Cailaud et al., Eur. J. Neurosci.5:1287-1291 (1993); Vincent et al., Nat. Genet. 5:130-134 (1993); Jaffeet al., Nat. Genet. 1:372-378 (1992); and Levrero et al., Gene101:195-202 (1992). Exemplary adenoviral gene therapy vectors employablein this invention also include those described in WO 94/12649, WO93/03769; WO 93/19191; WO 94/28938; WO95/11984 and WO 95/00655.Administration of DNA linked to killed adenovirus as described inCuriel, Hum. Gene Ther. 3:147-154 (1992) may be employed.

[0130] Other gene delivery vehicles and methods may be employed,including polycationic condensed DNA linked or unlinked to killedadenovirus alone, for example Curiel, Hum. Gene Ther. 3:147-154 (1992);ligand-linked DNA, for example see Wu, J. Biol. Chem. 264:16985-16987(1989); eukaryotic cell delivery vehicles cells, for example see U.S.Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796;deposition of photopolymerized hydrogel materials; hand-held genetransfer particle gun, as described in U.S. Pat. No. 5,149,655; ionizingradiation as described in U.S. Pat. No. 5,206,152 and in WO 92/11033;nucleic charge neutralization or fusion with cell membranes. Additionalapproaches are described in Philip, Mol. Cell Biol. 14:2411-2418 (1994),and in Woffendin, Proc. Natl. Acad Sci. 91:1581-1585 (1994).

[0131] Naked DNA may also be employed. Exemplary naked DNA introductionmethods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptakeefficiency may be improved using biodegradable latex beads. DNA coatedlatex beads are efficiently transported into cells after endocytosisinitiation by the beads. The method may be improved further by treatmentof the beads to increase hydrophobicity and thereby facilitatedisruption of the endosome and release of the DNA into the cytoplasm.Liposomes that can act as gene delivery vehicles are described in U.S.Pat. No. 5,422,120, PCT Patent Publication Nos. WO 95/13796, WO94/23697, and WO 91/14445, and EP No. 0 524 968.

[0132] Further non-viral delivery suitable for use includes mechanicaldelivery systems such as the approach described in Woffendin et al.,Proc. Natl. Acad. Sci. USA 91(24):11581-11585 (1994). Moreover, thecoding sequence and the product of expression of such can be deliveredthrough deposition of photopolymerized hydrogel materials. Otherconventional methods for gene delivery that can be used for delivery ofthe coding sequence include, for example, use of hand-held gene transferparticle gun, as described in U.S. Pat. No. 5,149,655; use of ionizingradiation for activating transferred gene, as described in U.S. Pat. No.5,206,152 and PCT Patent Publication No. WO 92/11033.

[0133] FGF has been implicated in diseases characterized by loss offunction, inadequate function/number, abnormal function or death ofcells, tissues or organs for which function or survival can beprolonged/rescued, and abnormalities reversed or prevented by therapywith FGF.

[0134] Loss of pulmonary, bronchia or alveolar cells or function,healing of pulmonary or bronchia wounds, pulmonary infraction,emphysema/chronic obstructive pulmonary disease, asthma, sequelae ofinfectious or autoimmune disease, sequelae of pulmonary arterial orvenous hypertension, pulmonary fibrosis, pulmonary disease ofimmaturity, and cystic fibrosis are conditions amenable to treatmentwith FGF.

[0135] Ischemic vascular disease may be amenable to FGF-21 treatment,wherein the disease is characterized by inadequate blood flow to anorgan(s). Treatment may induce therapeutic angiogenesis or preservefunction/survival of cells (myocardial ischemia/infarction, peripheralvascular disease, renal artery disease, stroke). Cardiomyopathiescharacterized by loss of function or death of cardiac myocytes orsupporting cells in the heart (congestive heart failure, myocarditis)may also be treated using FGF-21, as can musculoskeletal diseasecharacterized by loss of function, inadequate function or death ofskeletal muscle cells, bone cells or supporting cells. Examples includeskeletal myopathies, bone disease, and arthritis.

[0136] FGF-21 polynucleotides and polypeptides may aid in correction ofcongenital defects due to loss of FGF-21 molecule or its function(liver, heart, lung, brain, limbs, kidney, etc.).

[0137] Treatment of wound healing is yet another use of FGF-21polypeptides and polynucleotides, either due to trauma, disease, medicalor surgical treatment, including regeneration of cell populations andtissues depleted by these processes. Examples include liverregeneration, operative wound healing, re-endothelialization of injuredblood vessels, healing of traumatic wounds, healing of ulcers due tovascular, metabolic disease, etc., bone fractures, loss of cells due toinflammatory disease, etc.

[0138] FGF-21 may also be used in screens to identify drugs fortreatment of cancers which involve over activity of the molecule, or newtargets which would be useful in the identification of new drugs.

[0139] For all of the preceding embodiments, the clinician willdetermine, based on the specific condition, whether FGF-21 polypeptidesor polynucleotides, antibodies to FGF-21, or small molecules such aspeptide analogues or antagonists, will be the most suitable form oftreatment. These forms are all within the scope of the invention.

[0140] Preferred embodiments of the invention are described in thefollowing examples. Other embodiments within the scope of the claimsherein will be apparent to one skilled in the art from consideration ofthe specification or practice of the invention as disclosed herein. Itis intended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLES Example 1

[0141] Isolation and Analysis of Mouse FGF-21

[0142] DNA was prepared from mouse embryo cDNA. DNA was amplified frommouse embryo cDNA by polymerase chain reaction (PCR) for 30 cycles in 25μl of a reaction mixture containing each of the sense and antisensedegenerate primers representing all possible codons corresponding to theamino acid sequences of human FGF-19, RPYDGYN and LPMLPM, respectively.The amplified product was further amplified by PCR with each of thesense and antisense degenerate primers representing all possible codonscorresponding to the amino acid sequences of human FGF-19, RPDGYN andHFLPML, respectively. The amplified DNAs of expected size (approximately120 base pairs) were cloned. By determination of the nucleotidesequences of the cloned DNAs, a novel mouse FGF, FGF-21, cDNA wasidentified. To determine the entire coding region of the novel FGF cDNA,the coding region was amplified from mouse embryo cDNA byadaptor-ligation mediated PCR using a Marathon cDNA amplification kit(Clontech, Palo Alto, Calif.) and primers specific for the FGF. The cDNAencoding the entire coding region of the FGF was amplified from mouseembryo cDNA by PCR using the FGF-specific primers including the 5′ and3′ noncoding sequences, and cloned into the pGEM-T DNA vector. Thenucleotide sequence is shown in SEQ ID NO: 1 and the amino acid sequenceis shown in SEQ ID NO:2.

Example 2

[0143] Expression of FGF-21 in Mouse Tissues

[0144] Poly (A)+RNA (10 μg) from mouse tissues was dissolved on adenaturing agarose gel (1%) containing formaldehyde, and transferred toa nitrocellulose membrane in 20× SSC (IX SSC:0.15 M NACl/0.015 M sodiumcitrate) overnight. A ³²P-labeled FGF-21 cDNA probe (˜650 base pairs)was labeled with a random primer labeling kit (Pharmacia Biotech,Uppsala, Sweden) and deoxycytidine 5′-[α-³²P-] triphosphate (˜110TBq/mmol) (ICN Biomedicals Inc., Costa Mesa, Calif.). The membrane wasincubated in hybridization solution containing the labeled probe asdescribed (Hoshikawa et al., Biochem. Biophys. Res. Commun. 244:187-191(1998)), and analyzed with a radio-imaging analyzer (BAS 2000, FujiPhoto Film Co., Tokyo, Japan). As shown in FIG. 3, FGF-21 expression wasmost predominant in liver, with expression also seen in testis andthymus.

Example 3

[0145] Isolation and Analysis of Human FGF-21

[0146] The human FGF-21 gene was located in the 5′ flanking region of aputative human alpha 1,2-fucosyltransferase gene. The cDNA encoding theentire coding region of human FGF-21 was amplified from fetal brain cDNAby PCR using the FGF-specific primers including the 5′ and 3′ noncodingsequences, and cloned into the pGEM-T DNA vector. The protein contains209 amino acids, as shown in SEQ ID NO:4 (FIG. 5), and is encoded by thepolynucleotide sequence of SEQ ID NO:3. Primers for amplification ofhuman FGF-21 cDNA coding region are: sense primer for FGF-21: 5′agccattgatggactcggac 3′; antisense primer for FGF-21: 5′tggcttcaggaagcgtagct 3′.

Example 4

[0147] Preparation of Antisera to FGF-21 by Immunization of Rabbits withan FGF-21 Peptide

[0148] A peptide sequence corresponding to selected contiguous aminoacids of the human FGF-21 protein is synthesized and coupled to keyholelimpet hemocyanin (KLH) as described (Harlow and Land, Antibodies: ALaboratory Manual, 1988. Cold Spring Harbor Laboratory, New York, N.Y.)The KLH-coupled peptide is used to immunize rabbits. Antisera are testedfor specificity to FGF-21, and for cross-reactivity with other FGFproteins.

[0149] Exemplary peptide sequences are: 1. RQRYLYDDAQQTEAH (residues46-61 of SEQ ID NO:4) 2. HLPGNKSPHRDPAPR (residues 146-160 of SEQ ID NO:4)

[0150] All patents, published patent applications and publications citedherein are incorporated by reference as if set forth fully herein.

[0151] Although certain preferred embodiments have been describedherein, it is not intended that such embodiments be construed aslimitations on the scope of the invention except as set forth in thefollowing claims.

1 17 1 659 DNA Mus musculus CDS (14)...(646) 1 gagcgcagcc ctg atg gaatgg atg aga tct aga gtt ggg acc ctg gga 49 Met Glu Trp Met Arg Ser ArgVal Gly Thr Leu Gly 1 5 10 ctg tgg gtc cga ctg ctg ctg gct gtc ttc ctgctg ggg gtc tac caa 97 Leu Trp Val Arg Leu Leu Leu Ala Val Phe Leu LeuGly Val Tyr Gln 15 20 25 gca tac ccc atc cct gac tcc agc ccc ctc ctc cagttt ggg ggt caa 145 Ala Tyr Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln PheGly Gly Gln 30 35 40 gtc cgg cag agg tac ctc tac aca gat gac gac caa gacact gaa gcc 193 Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Asp Gln Asp ThrGlu Ala 45 50 55 60 cac ctg gag atc agg gag gat gga aca gtg gta ggc gcagca cac cgc 241 His Leu Glu Ile Arg Glu Asp Gly Thr Val Val Gly Ala AlaHis Arg 65 70 75 agt cca gaa agt ctc ctg gag ctc aaa gcc ttg aag cca ggggtc att 289 Ser Pro Glu Ser Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly ValIle 80 85 90 caa atc ctg ggt gtc aaa gcc tct agg ttt ctt tgc caa cag ccagat 337 Gln Ile Leu Gly Val Lys Ala Ser Arg Phe Leu Cys Gln Gln Pro Asp95 100 105 gga gct ctc tat gga tcg cct cac ttt gat cct gag gcc tgc agcttc 385 Gly Ala Leu Tyr Gly Ser Pro His Phe Asp Pro Glu Ala Cys Ser Phe110 115 120 aga gaa ctg ctg ctg gag gac ggt tac aat gtg tac cag tct gaagcc 433 Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala125 130 135 140 cat ggc ctg ccc ctg cgt ctg cct cag aag gac tcc cca aaccag gat 481 His Gly Leu Pro Leu Arg Leu Pro Gln Lys Asp Ser Pro Asn GlnAsp 145 150 155 gca aca tcc tgg gga cct gtg cgc ttc ctg ccc atg cca ggcctg ctc 529 Ala Thr Ser Trp Gly Pro Val Arg Phe Leu Pro Met Pro Gly LeuLeu 160 165 170 cac gag ccc caa gac caa gca gga ttc ctg ccc cca gag ccccca gat 577 His Glu Pro Gln Asp Gln Ala Gly Phe Leu Pro Pro Glu Pro ProAsp 175 180 185 gtg ggc tcc tct gac ccc ctg agc atg gta gag cct tta cagggc cga 625 Val Gly Ser Ser Asp Pro Leu Ser Met Val Glu Pro Leu Gln GlyArg 190 195 200 agc ccc agc tat gcg tcc tga ctcttcctga atc 659 Ser ProSer Tyr Ala Ser * 205 210 2 210 PRT Mus musculus 2 Met Glu Trp Met ArgSer Arg Val Gly Thr Leu Gly Leu Trp Val Arg 1 5 10 15 Leu Leu Leu AlaVal Phe Leu Leu Gly Val Tyr Gln Ala Tyr Pro Ile 20 25 30 Pro Asp Ser SerPro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg 35 40 45 Tyr Leu Tyr ThrAsp Asp Asp Gln Asp Thr Glu Ala His Leu Glu Ile 50 55 60 Arg Glu Asp GlyThr Val Val Gly Ala Ala His Arg Ser Pro Glu Ser 65 70 75 80 Leu Leu GluLeu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly 85 90 95 Val Lys AlaSer Arg Phe Leu Cys Gln Gln Pro Asp Gly Ala Leu Tyr 100 105 110 Gly SerPro His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu 115 120 125 LeuGlu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro 130 135 140Leu Arg Leu Pro Gln Lys Asp Ser Pro Asn Gln Asp Ala Thr Ser Trp 145 150155 160 Gly Pro Val Arg Phe Leu Pro Met Pro Gly Leu Leu His Glu Pro Gln165 170 175 Asp Gln Ala Gly Phe Leu Pro Pro Glu Pro Pro Asp Val Gly SerSer 180 185 190 Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser ProSer Tyr 195 200 205 Ala Ser 210 3 643 DNA Homo sapiens CDS (9)...(638) 3agccattg atg gac tcg gac gag acc ggg ttc gag cac tca gga ctg tgg 50 MetAsp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp 1 5 10 gtt tct gtgctg gct ggt ctt ctg ctg gga gcc tgc cag gca cac ccc 98 Val Ser Val LeuAla Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro 15 20 25 30 atc cct gactcc agt cct ctc ctg caa ttc ggg ggc caa gtc cgg cag 146 Ile Pro Asp SerSer Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln 35 40 45 cgg tac ctc tacaca gat gat gcc cag cag aca gaa gcc cac ctg gag 194 Arg Tyr Leu Tyr ThrAsp Asp Ala Gln Gln Thr Glu Ala His Leu Glu 50 55 60 atc agg gag gat gggacg gtg ggg ggc gct gct gac cag agc ccc gaa 242 Ile Arg Glu Asp Gly ThrVal Gly Gly Ala Ala Asp Gln Ser Pro Glu 65 70 75 agt ctc ctg cag ctg aaagcc ttg aag ccg gga gtt att caa atc ttg 290 Ser Leu Leu Gln Leu Lys AlaLeu Lys Pro Gly Val Ile Gln Ile Leu 80 85 90 gga gtc aag aca tcc agg ttcctg tgc cag cgg cca gat ggg gcc ctg 338 Gly Val Lys Thr Ser Arg Phe LeuCys Gln Arg Pro Asp Gly Ala Leu 95 100 105 110 tat gga tcg ctc cac tttgac cct gag gcc tgc agc ttc cgg gag ctg 386 Tyr Gly Ser Leu His Phe AspPro Glu Ala Cys Ser Phe Arg Glu Leu 115 120 125 ctt ctt gag gac gga tacaat gtt tac cag tcc gaa gcc cac ggc ctc 434 Leu Leu Glu Asp Gly Tyr AsnVal Tyr Gln Ser Glu Ala His Gly Leu 130 135 140 ccg ctg cac ctg cca gggaac aag tcc cca cac cgg gac cct gca ccc 482 Pro Leu His Leu Pro Gly AsnLys Ser Pro His Arg Asp Pro Ala Pro 145 150 155 cga gga cca gct cgc ttcctg cca cta cca ggc ctg ccc ccc gca ctc 530 Arg Gly Pro Ala Arg Phe LeuPro Leu Pro Gly Leu Pro Pro Ala Leu 160 165 170 ccg gag cca ccc gga atcctg gcc ccc cag ccc ccc gat gtg ggc tcc 578 Pro Glu Pro Pro Gly Ile LeuAla Pro Gln Pro Pro Asp Val Gly Ser 175 180 185 190 tcg gac cct ctg agcatg gtg gga cct tcc cag ggc cga agc ccc agc 626 Ser Asp Pro Leu Ser MetVal Gly Pro Ser Gln Gly Arg Ser Pro Ser 195 200 205 tac gct tcc tgaagcca 643 Tyr Ala Ser * 4 209 PRT Homo sapiens 4 Met Asp Ser Asp Glu ThrGly Phe Glu His Ser Gly Leu Trp Val Ser 1 5 10 15 Val Leu Ala Gly LeuLeu Leu Gly Ala Cys Gln Ala His Pro Ile Pro 20 25 30 Asp Ser Ser Pro LeuLeu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr 35 40 45 Leu Tyr Thr Asp AspAla Gln Gln Thr Glu Ala His Leu Glu Ile Arg 50 55 60 Glu Asp Gly Thr ValGly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu 65 70 75 80 Leu Gln Leu LysAla Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val 85 90 95 Lys Thr Ser ArgPhe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly 100 105 110 Ser Leu HisPhe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu 115 120 125 Glu AspGly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu 130 135 140 HisLeu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly 145 150 155160 Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu 165170 175 Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp180 185 190 Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser TyrAla 195 200 205 Ser 5 20 DNA Artificial Sequence PCR primer 5 agccattgatggactcggac 20 6 20 DNA Artificial Sequence PCR primer 6 tggcttcaggaagcgtagct 20 7 16 PRT Homo sapiens 7 Arg Gln Arg Tyr Leu Tyr Thr AspAsp Ala Gln Gln Thr Glu Ala His 1 5 10 15 8 15 PRT Homo sapiens 8 HisLeu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg 1 5 10 15 9 218PRT Mus musculus 9 Met Ala Arg Lys Trp Asn Gly Arg Ala Val Ala Arg AlaLeu Val Leu 1 5 10 15 Ala Thr Leu Trp Leu Ala Val Ser Gly Arg Pro LeuAla Gln Gln Ser 20 25 30 Gln Ser Val Ser Asp Glu Asp Pro Leu Phe Leu TyrGly Trp Gly Lys 35 40 45 Ile Thr Arg Leu Gln Tyr Leu Tyr Ser Ala Gly ProTyr Val Ser Asn 50 55 60 Cys Phe Leu Arg Ile Arg Ser Asp Gly Ser Val AspCys Glu Glu Asp 65 70 75 80 Gln Asn Glu Arg Asn Leu Leu Glu Phe Arg AlaVal Ala Leu Lys Thr 85 90 95 Ile Ala Ile Lys Asp Val Ser Ser Val Arg TyrLeu Cys Met Ser Ala 100 105 110 Asp Gly Lys Ile Tyr Gly Leu Ile Arg TyrSer Glu Glu Asp Cys Thr 115 120 125 Phe Arg Glu Glu Met Asp Cys Leu GlyTyr Asn Gln Tyr Arg Ser Met 130 135 140 Lys His His Leu His Ile Ile PheIle Gln Ala Lys Pro Arg Glu Gln 145 150 155 160 Leu Gln Asp Gln Lys ProSer Asn Phe Ile Pro Val Phe His Arg Ser 165 170 175 Phe Phe Glu Thr GlyAsp Gln Leu Arg Ser Lys Met Phe Ser Leu Pro 180 185 190 Leu Glu Ser AspSer Met Asp Pro Phe Arg Met Val Glu Asp Val Asp 195 200 205 His Leu ValLys Ser Pro Ser Phe Gln Lys 210 215 10 216 PRT Homo sapiens 10 Met ArgSer Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 TrpLeu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 HisVal His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35 40 45 ThrSer Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala 50 55 60 AspGly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu 65 70 75 80Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val His 85 90 95Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly Leu 100 105110 Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro 115120 125 Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser130 135 140 Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly PheLeu 145 150 155 160 Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val ProGlu Glu Pro 165 170 175 Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met PheSer Ser Pro Leu 180 185 190 Glu Thr Asp Ser Met Asp Pro Phe Gly Leu ValThr Gly Leu Glu Ala 195 200 205 Val Arg Ser Pro Ser Phe Glu Lys 210 21511 10 PRT Unknown Residues which contain the anitgenic determinantrecognized by the myc monoclonal antibody. 11 Glu Gln Lys Leu Ile SerGlu Glu Asp Leu 1 5 10 12 5 PRT Unknown Preferred thrombin cleave site.12 Leu Val Pro Arg Gly 1 5 13 10 PRT Unknown Residues which bind toparamagnetic streptavidin beads (used for purification). 13 Ser Ala TrpArg His Pro Gln Phe Gly Gly 1 5 10 14 7 PRT Homo sapiens 14 Arg Pro TyrAsp Gly Tyr Asn 1 5 15 6 PRT Homo sapiens 15 Leu Pro Met Leu Pro Met 1 516 6 PRT Homo sapiens 16 Arg Pro Asp Gly Tyr Asn 1 5 17 6 PRT Homosapiens 17 His Phe Leu Pro Met Leu 1 5

We claim:
 1. An isolated nucleic acid molecule comprising apolynucleotide selected from the group consisting of: (a) apolynucleotide encoding amino acids from about 1 to about 209 of SEQ IDNO:4; (b) a polynucleotide encoding amino acids from about 2 to about209 of SEQ ID NO:4; (c) a polynucleotide encoding amino acids from about1 to about 177 of SEQ ID NO:4; (d) a polynucleotide encoding amino acidsfrom about 40 to about 209 of SEQ ID NO:4; (e) a polynucleotide encodingamino acids from about 40 to about 177 of SEQ ID NO:4; (f) thepolynucleotide complement of (a), (b), (c), (d) or (e); and (g) apolynucleotide at least 90% identical to the polynucleotide of (a), (b),(c), (d) or (e).
 2. An isolated nucleic acid molecule which comprises20-600 contiguous nucleotides from the coding region of SEQ ID NO:3. 3.The isolated nucleic acid molecule of claim 2, which comprises 60-400contiguous nucleotides from the coding region of SEQ ID NO:3
 4. Theisolated nucleic acid molecule of claim 3, which comprises 200-300contiguous nucleotides from the coding region of SEQ ID NO:3.
 5. Anisolated nucleic acid molecule comprising a polynucleotide encoding apolypeptide wherein, except for at least one conservative amino acidsubstitution, said polypeptide has an amino acid sequence selected fromthe group consisting of: (a) amino acids from about 1 to about 209 ofSEQ ID NO:4; (b) amino acids from about 2 to about 209 of SEQ ID NO:4;(c) amino acids from about 1 to about 177 of SEQ ID NO:4; (d) aminoacids from about 40 to about 209 of SEQ ID NO:4; and (e) amino acidsfrom about 40 to about 177 of SEQ ID NO:4.
 6. The isolated nucleic acidmolecule of claim 1, which is DNA.
 7. A method of making a recombinantvector comprising inserting a nucleic acid molecule of claim 1 into avector in operable linkage to a promoter.
 8. A recombinant vectorproduced by the method of claim
 7. 9. A method of making a recombinanthost cell comprising introducing the recombinant vector of claim 8 intoa host cell.
 10. A recombinant host cell produced by the method of claim9.
 11. A recombinant method of producing a polypeptide, comprisingculturing the recombinant host cell of claim 10 under conditions suchthat said polypeptide is expressed and recovering said polypeptide. 12.An isolated polypeptide comprising amino acids at least 95% identical toamino acids selected from the group consisting of: (a) amino acids fromabout 1 to about 209 of SEQ ID NO:4; (b) amino acids from about 2 toabout 209 of SEQ ID NO:4; (c) amino acids from about 1 to about 177 ofSEQ ID NO:4; (d) amino acids from about 40 to about 209 of SEQ ID NO:4;and (e) amino acids from about 40 to about 177 of SEQ ID NO:4.
 13. Anisolated polypeptide wherein, except for at least one conservative aminoacid substitution, said polypeptide has an amino acid sequence selectedfrom the group consisting of: (a) amino acids from about 1 to about 209of SEQ ID NO:4; (b) amino acids from about 2 to about 209 of SEQ IDNO:4; (c) amino acids from about 1 to about 177 of SEQ ID NO:4; (d)amino acids from about 40 to about 209 of SEQ ID NO:4; and (e) aminoacids from about 40 to about 177 of SEQ ID NO:4.
 14. An isolatedpolypeptide comprising amino acids selected from the group consistingof: (a) amino acids from about 1 to about 209 of SEQ ID NO:4; (b) aminoacids from about 2 to about 209 of SEQ ID NO:4; (c) amino acids fromabout 1 to about 177 of SEQ ID NO:4; (d) amino acids from about 40 toabout 209 of SEQ ID NO:4; and (e) amino acids from about 40 to about 177of SEQ ID NO:4.
 15. An epitope-bearing portion of the polypeptide of SEQID NO:4.
 16. The epitope-bearing portion of claim 15, which comprisesbetween 10 and 50 contiguous amino acids of SEQ ID NO:4.
 17. Theepitope-bearing portion of claim 15, which comprises amino acidsRQRYLYTDDAQQTEAH (SEQ ID NO:7).
 18. The epitope-bearing portion of claim15, which comprises amino acids HLPGNKSPHRDPAPR (SEQ ID NO:8).
 19. Anisolated antibody that binds specifically to the polypeptide of claim12.
 20. An isolated antibody that binds specifically to the polypeptideof claim
 13. 21. An isolated antibody that binds specifically to thepolypeptide of claim
 14. 22. A pharmaceutical composition comprising thepolypeptide of claim 12, in combination with a pharmaceuticallyacceptable carrier.
 23. A method for providing trophic support for cellsin a patient in need thereof, the method comprising administering to thepatient a composition comprising a polynucleotide encoding thepolypeptide of SEQ ID NO:4.
 24. The method of claim 23 wherein saidpolynucleotide is administered by implanting cells which express saidpolynucleotide into the patient, wherein said cells express FGF-21polypeptide in the patient.
 25. The method of claim 23 wherein theimplanted cells are encapsulated in a semipermeable membrane.
 26. Themethod of claim 23 wherein the patient suffers from a conditioncharacterized by inadequate numbers of hepatic cells.
 27. The method ofclaim 23 wherein the condition is cirrhosis of the liver.
 28. The methodof claim 23 wherein said patient suffers from a condition characterizedby inadequate function or number of testicular cells.
 29. The method ofclaim 28 wherein said condition is at least one condition selected fromthe group consisting of infertility, impotence, and testicular cancer.30. The method of claim 23 wherein the patient suffers from a conditioncharacterized by inadequate function of the thymus.
 31. The method ofclaim 30 wherein said condition is at least one condition selected fromthe group consisting of leukemia, lymphoma, autoimmune disease,proliferative disorder of the thymus, and differentiation disorder ofthe thymus.
 32. A method for providing trophic support for cells in apatient in need thereof, the method comprising administering to thepatient a composition comprising a polypeptide of SEQ ID NO:4.
 33. Themethod of claim 28 wherein the patient suffers from a conditioncharacterized by inadequate numbers of hepatic cells.
 34. The method ofclaim 29 wherein the condition is cirrhosis of the liver.
 35. A methodof alleviating a disease condition in the liver of a human patientwherein said disease condition is alleviated by at least one methodselected from the group consisting of slowing degeneration of, restoringfunction of, and increasing the number of, functional hepatic cells insaid human patient, said method comprising administering to said patienta pharmaceutically effective composition comprising a polypeptide havingthe amino acid sequence of SEQ ID NO:4.
 36. A method of alleviating adisease condition in the thymus of a human patient wherein said diseasecondition is alleviated by at least one method selected from the groupconsisting of preventing degeneration of, slowing degeneration of,increasing the number of, functional thymic cells in said human patient,said method comprising administering to said patient a pharmaceuticallyeffective composition comprising a polypeptide having the amino acidsequence of SEQ ID NO:4.
 37. A method of alleviating a disease conditionin the testis of a human patient wherein said disease condition isalleviated by at least one method selected from the group consisting ofpreventing degeneration of, slowing degeneration of, and increasing thenumber of, functional testicular cells in said human patient, saidmethod comprising administering to said patient a pharmaceuticallyeffective composition comprising a polypeptide having the amino acidsequence of SEQ ID NO:4.
 38. A kit for detecting the presence of mRNAencoding FGF-21 in a sample from a patient, said kit comprising apolynucleotide having at least 20 contiguous nucleotides of thepolynucleotide of claim 3, packaged in a container.
 39. The kitaccording to claim 38 wherein the polynucleotide encodes at least sixcontiguous amino acids of SEQ ID NO:4.
 40. A kit for detecting thepresence of FGF-21 polypeptide in a sample from a patient, said kitcomprising an antibody according to claim 19, packaged in a container.41. An isolated nucleic acid molecule comprising a polynucleotideselected from the group consisting of: (a) a polynucleotide encodingamino acids from about 1 to about 210 of SEQ ID NO:2; (b) apolynucleotide encoding amino acids from about 2 to about 21 of SEQ IDNO:2; (c) a polynucleotide encoding amino acids from about 1 to about177 of SEQ ID NO:2; (d) a polynucleotide encoding amino acids from about40 to about 210 of SEQ ID NO:2; (e) a polynucleotide encoding aminoacids from about 40 to about 177 of SEQ ID NO:2; (f) the polynucleotidecomplement of (a), (b), (c), (d) or (e); and (h) a polynucleotide atleast 90% identical to the polynucleotide of (a), (b), (c), (d) or (e).42. An isolated nucleic acid molecule which comprises 20-600 contiguousnucleotides from the coding region of SEQ ID NO:
 1. 43. The isolatednucleic acid molecule of claim 42, which comprises 60-400 contiguousnucleotides from the coding region of SEQ ID NO:
 1. 44. The isolatednucleic acid molecule of claim 43, which comprises 200-300 contiguousnucleotides from the coding region of SEQ ID NO:
 1. 45. An isolatednucleic acid molecule comprising a polynucleotide encoding a polypeptidewherein, except for at least one conservative amino acid substitution,said polypeptide has an amino acid sequence selected from the groupconsisting of: (a) amino acids from about 1 to about 210 of SEQ ID NO:2;(b) amino acids from about 2 to about 210 of SEQ ID NO:2; (c) aminoacids from about 1 to about 177 of SEQ ID NO:2; (d) amino acids fromabout 40 to about 210 of SEQ ID NO:2; and (e) amino acids from about 40to about 177 of SEQ ID NO:4.
 46. The isolated nucleic acid molecule ofclaim 41, which is DNA.
 47. A method of making a recombinant vectorcomprising inserting a nucleic acid molecule of claim 41 into a vectorin operable linkage to a promoter.
 48. A recombinant vector produced bythe method of claim
 47. 49. A method of making a recombinant host cellcomprising introducing the recombinant vector of claim 48 into a hostcell.
 50. A recombinant host cell produced by the method of claim 49.51. A recombinant method of producing a polypeptide, comprisingculturing the recombinant host cell of claim 50 under conditions suchthat said polypeptide is expressed and recovering said polypeptide. 52.An isolated polypeptide comprising amino acids at least 95% identical toamino acids selected from the group consisting of: (a) amino acids fromabout 1 to about 210 of SEQ ID NO:2; (b) amino acids from about 2 toabout 210 of SEQ ID NO:2; (c) amino acids from about 1 to about 177 ofSEQ ID NO:2; (d) amino acids from about 40 to about 210 of SEQ ID NO:2;and (e) amino acids from about 40 to about 177 of SEQ ID NO:2.
 53. Anisolated polypeptide wherein, except for at least one conservative aminoacid substitution, said polypeptide has an amino acid sequence selectedfrom the group consisting of: (a) amino acids from about 1 to about 210of SEQ ID NO:2; (b) amino acids from about 2 to about 210 of SEQ IDNO:2; (c) amino acids from about 1 to about 177 of SEQ ID NO:2; (d)amino acids from about 40 to about 210 of SEQ ID NO:2; and (e) aminoacids from about 40 to about 177 of SEQ ID NO:2.
 54. An isolatedpolypeptide comprising amino acids selected from the group consistingof: (a) amino acids from about 1 to about 210 of SEQ ID NO:2; (b) aminoacids from about 2 to about 210 of SEQ ID NO:2; (c) amino acids fromabout 1 to about 177 of SEQ ID NO:2; (d) amino acids from about 40 toabout 210 of SEQ ID NO:2; and (e) amino acids from about 40 to about 177of SEQ ID NO:2.
 55. An epitope-bearing portion of the polypeptide of SEQID NO:2.
 56. The epitope-bearing portion of claim 55, which comprisesbetween 10 and 50 contiguous amino acids of SEQ ID NO:2.
 57. An isolatedantibody that binds specifically to the polypeptide of claim
 52. 58. Anisolated antibody that binds specifically to the polypeptide of claim53.
 59. An isolated antibody that binds specifically to the polypeptideof claim 54.